A number of techniques are known and have been developed for detecting the presence of nucleic acids and their structural changes. However, most of these techniques chemically alter the nucleic acid of interest or change the molecule in some fashion. For instance, modification of DNA sequences has been accomplished using restriction enzymes. These enzymes digest the nucleic acid into defined sequences and alter the defined order or continuity of the molecule. Restriction enzymes have been used in diagnostic medicine and molecular biology as well as genomics, genotyping, DNA diagnostics, molecular diagnostics and high throughput screening.
More recently, solutions to hybridization detection use labeled probes and/or targets that provide various optical or electrical signals when probes and targets hybridize. This type of technique has significant advantages over the above-described techniques that require complete chemical modification.
Other improved methods that use partial chemical modification are also known in the art. For instance, U.S. Pat. No. 5,591,578 to Meade et al., teaches the application of ruthenium complexes to the backbone of nucleic acid molecules. The ruthenium complexes are covalently bound to the ribose-phosphate backbone of the nucleic acid at predetermined positions. In addition, Lee (WO 99/31115 and Biochem. Cell Biol. Vol. 71, 1993, 162–168) teaches a number of techniques for hybridization detection using the electrical properties of M-DNA. However, each of these techniques, as well as others described in the literature, require modification of the probe as well as the sample used in the detection. In particular, the method of Lee et al. requires the use of more than one tag that has been chemically attached to the probe and/or target DNA for fluorescence detection. The need for the modification of the sample DNA is a common step for several of the hybridization detection techniques. The techniques require extensive sample preparation to obtain and modify DNA that works with these types of tests. Other disadvantages with the prior art concern detecting single base pair mismatches in the DNA after hybridization. In order to be able to detect such mismatches, very stringent conditions of pH, temperature and salt concentration must be maintained. (Jonathan A. Prince, Lars, Feuk, W. Mathias Howell, Magnus Jobs, Tesfai Emahazion, Kaj Blennow and Anthony J. Brookes, Genome Research, 11, 2001, 152–162) This often leads to high error rate in the detection of single base mismatches as in single nucleotide polymorphism (SNP) scoring.
Accordingly, there is an ongoing need for new inventions, methods and techniques that provide high signal and sensitivity during or after the hybridization process. These techniques should also have broad application to high throughput screening and microarray platforms. Furthermore, there is a need for a technique that is simple to use, needs minimal sample preparation and is capable of detecting single base pair mismatches using a simple hybridization procedure.